Pediococcus pentosaceus MDL511 and application thereof in preparation of hypolipidemic products

Abstract

The invention discloses a pediococcus pentosaceus MDL511, which is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.35201, the preservation date is July 11, 2025, and the classification name of the strain is pediococcus pentosaceus. The pediococcus pentosaceus MDL511 has the advantages that the preservation number is CGMCC No.35201, the preservation number is CGMCC No.35201, the preservation date is July 11, 2025, and the classification name of the strain is pediococcus pentosaceus. The pediococcus pentosaceus MDL511 disclosed by the invention can be used for remarkably reducing weight gain and fat growth of mice caused by high fat diet, reducing serum cholesterol, LDL-C (Low Density Lipoprotein Cholesterol) and ALT (Atomic Lymphocyte Transferase) levels and remarkably improving hepatic lesions and fat accumulation caused by high fat diet, so that the MDL511 has a blood fat reducing effect, can be used for preparing blood fat reducing products and weight losing products and has a good development prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to Pediococcus pentosaceus MDL511 and its application in the preparation of lipid-lowering products. Background Technology

[0002] High blood lipids can accelerate atherosclerosis and increase the risk of cardiovascular and cerebrovascular diseases. Although existing chemical drugs can control the indicators, they have problems such as significant side effects, poor adherence, and rebound effects after discontinuation.

[0003] Probiotics are a class of microecological regulators for the human body, capable of improving gut microbiota structure, promoting the proliferation of beneficial bacteria, inhibiting the growth of harmful bacteria, eliminating pathogenic factors, and enhancing immunity. Certain probiotics can help lower blood lipids through mechanisms such as producing short-chain fatty acids, regulating inflammatory signaling pathways, and improving intestinal barrier function, making them clinically significant for the prevention and treatment of diseases such as hyperlipidemia and heart disease. However, different strains exhibit significant functional differences, and existing strains often fall short in terms of acid and bile salt tolerance, colonization ability, and metabolic activity. Therefore, it is necessary to develop new lipid-lowering strains to provide new research directions for addressing obesity caused by high cholesterol and improving gut microbiota. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to provide a lipid-lowering Pediococcus pentosaceus MDL511, a lipid-lowering bacterial agent, and its application in the preparation of lipid-lowering products.

[0005] The technical solution adopted in this invention is: *Pediococcus pentosaceus* MDL511, deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 35201, deposit date July 11, 2025, and the strain is classified and named as *Pediococcus pentosaceus* (…). Pediococcus pentosaceus ).

[0006] The present invention also provides a Pediococcus pentosaceus MDL511 inoculum, the active ingredient of which comprises fermented cells of Pediococcus pentosaceus MDL511.

[0007] Furthermore, the Pediococcus pentosaceus MDL511 inoculum is a lyophilized powder.

[0008] In the Pediococcus pentosaceus MDL511 inoculum, the viable count of Pediococcus pentosaceus MDL511 is not less than 5 × 10⁻⁶. 10 CFU / mL or 2×10 11 CFU / g.

[0009] Furthermore, the Pediococcus pentosaceus MDL511 inoculum agent also includes a protectant and / or functional adjuvant; The protective agent includes one or more of the following: skim milk powder, gelatin, dextrin, gum arabic, dextran, sodium alginate, polyvinylpyrrolidone, sucrose, lactose, trehalose, sorbitol, xylitol, maltodextrin, fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, polydextrose, and monosodium glutamate.

[0010] The fermentation cells of Pediococcus pentosaceus MDL511 are generally obtained by activating, seed culture, and fermentation culture of Pediococcus pentosaceus MDL511.

[0011] This invention also provides the application of Pediococcus pentosaceus MDL511 in the preparation of lipid-lowering products.

[0012] This invention also provides the application of Pediococcus pentosaceus MDL511 in the preparation of products that lower serum cholesterol, LDL-C, and ALT levels.

[0013] The present invention also provides the use of Pediococcus pentosaceus MDL511 in the preparation of products for the prevention, improvement or treatment of obesity.

[0014] This invention also provides the application of Pediococcus pentosaceus MDL511 in the preparation of products that reduce weight gain and fat accumulation caused by a high-fat diet.

[0015] The present invention also provides a lipid-lowering product, including Pediococcus pentosaceus MDL511 bacterial agent.

[0016] Furthermore, the lipid-lowering product can reduce serum cholesterol, LDL-C, and ALT levels.

[0017] The present invention also provides a weight loss product, comprising Pediococcus pentosaceus MDL511 bacterial agent.

[0018] Furthermore, the weight loss product can reduce weight gain and fat accumulation caused by a high-fat diet.

[0019] The lipid-lowering products mentioned can be lipid-lowering drugs or food.

[0020] The dosage forms of lipid-lowering products include common dosage forms in this field such as lyophilized powder, granules, capsules, tablets, powders, oral liquids, suspensions, or emulsions.

[0021] Lipid-lowering products may also include pharmaceutical excipients acceptable to the human body, such as fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, colorants, pH regulators, antioxidants, antibacterial agents, or buffers, etc.

[0022] The dosage of lipid-lowering products can be 10 10 CFU / day.

[0023] The *Pediococcus pentosaceus* MDL511, with accession number CGMCC No. 35201, described in this invention, was screened and isolated from shrimp samples. It belongs to the lactic acid bacteria (LAB) group and is a commensal bacterium of humans. It is Gram-positive, spherical, arranged in tetrads or pairs, and non-spore-forming; the strain has a diameter of 0.5-1.0 μm, lacks flagella, and does not form spores; exhibiting typical *Pediococcus pentosaceus* morphology.

[0024] The beneficial effects of this invention are as follows: the Pediococcus pentosus MDL511 of this invention can significantly reduce weight gain, fat growth, and decrease serum cholesterol, LDL-C and ALT levels in mice induced by a high-fat diet, and significantly improve liver lesions and fat accumulation caused by HFD. Therefore, MDL511 has a lipid-lowering effect and can be used to prepare lipid-lowering products, and has good development prospects.

[0025] The results of drug resistance gene analysis showed that when the whole genome sequencing results of MDL511 were compared with the CARD drug resistance gene database, the number of possible drug resistance genes matched was 0 when the Identity was greater than 80% and the Coverage was greater than 70%, meaning that there were no drug resistance genes.

[0026] Virulence gene analysis results showed that when the whole genome sequencing results of MDL511 were compared with the VFDB virulence gene database, the number of possible virulence genes matched was 0 when the Identity was greater than 80% and the Coverage was greater than 70%, meaning that no virulence genes were found.

[0027] Analysis of mobile elements revealed four gene islands in the MDL511 genome. However, no virulence or resistance genes were found, indicating an extremely low risk of horizontal metastasis.

[0028] In summary, whole-genome analysis revealed that this strain does not contain virulence genes or drug resistance genes, therefore MDL511 has high safety and great potential for clinical application. Attached Figure Description

[0029] Figure 1 The images show the morphological characteristics of the isolated strains after culture. The left image shows the morphological characteristics of the isolated strains on a plate, and the right image shows the morphological characteristics of the isolated strains under a microscope (1000×).

[0030] Figure 2 This is a 3000x magnified image of the MDL511 strain taken with a scanning electron microscope. Each small scale bar represents 1 μm.

[0031] Figure 3 This is a 20,000x magnified image of the MDL511 strain taken by transmission electron microscopy. The scale bar represents 500 nm.

[0032] Figure 4 This is the genome map of MDL511 after whole-genome sequencing.

[0033] Figure 5 Line graph showing the changes in body weight of mice in different groups.

[0034] Figure 6 A comparison chart showing the weight gain of mice in different groups.

[0035] Figure 7 The graph shows the statistical results of the percentage of epididymal fat, groin fat, and perirenal fat in mice from different groups.

[0036] Figure 8 The figure shows a comparison of the levels of four lipid parameters (TC, TG, HDL-C, and LDL-C) in mice from different groups.

[0037] Figure 9 A comparison of serum ALT and AST levels in mice from different groups.

[0038] Figure 10 HE staining images of epididymal adipose tissue from different groups of mice. In the images, each small grid represents 40 μm.

[0039] Figure 11 HE staining images of liver tissue from different groups of mice. In the image, each small grid represents 20 μm.

[0040] Figure 12 Oil Red O staining images of liver tissue from different groups of mice. In the image, each small grid represents 20 μm. Detailed Implementation

[0041] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the embodiments of this invention will be described in detail below with reference to the accompanying drawings to facilitate understanding by those skilled in the art. However, the scope of protection of this invention is not limited thereto.

[0042] Example 1

[0043] Pediococcus pentosaceus MDL511, deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 35201, deposited on July 11, 2025, and classified as Pediococcus pentosaceus (… Pediococcus pentosaceus ).

[0044] The specific screening and separation method is as follows: Take 100 μL of shrimp sample from the preservation tube and add it to an EP tube pre-filled with 900 μL of sterile PBS. Perform serial dilutions on the sample. Take samples of different dilutions, streak them on MRS solid medium, and place them in an incubator. Incubate at 37℃ and 0.5% CO2 for 48 h.

[0045] Remove the culture dish and use a sterile inoculation loop to pick colonies with different morphological characteristics. Transfer them to new MRS solid medium for purification and anaerobic culture at 37°C for 48 hours. Repeat the transfer three times. Culture the purified strain in liquid MRS at pH 3.5 and screen for strains with excellent acid tolerance. Finally, Pediococcus pentosaceus strain MDL511 was obtained.

[0046] MDL511 was inoculated onto MRS agar plates and cultured anaerobically (5% CO2 / 10% H2 / 85% N2) at 37℃±1℃ for 2-3 days. The colony morphology on the plates is as follows. Figure 1 As shown in the left image, the colonies are raised, round, smooth, moist, and white. Typical colonies grown on the culture medium are picked and observed under a microscope after Gram staining. Figure 1 The image on the right shows Gram-positive bacteria that are non-spore-forming, non-flagellated, non-motile, spherical, and arranged in tetrads or pairs.

[0047] A 3000x magnified image of the MDL511 strain taken with a scanning electron microscope, as shown below. Figure 2 As shown, the transmission electron microscope image of strain MDL511 is as follows. Figure 3 As shown. Scanning electron microscopy revealed that the cells were relatively uniform in size, with intact cell walls, no obvious shrinkage or collapse, and uniform surface mucus, without flagella, pili, spores, or other structures. Transmission electron microscopy showed typical Pediococcus pentosaceus morphology, without mycoplasma, virus-like particles, or other microbial contamination.

[0048] A single colony of this bacterium was picked and added to 10 mL of MRS liquid medium, anaerobically cultured overnight at 37°C, and then transported via cold chain to Sangon Biotech (Shanghai) Co., Ltd. for 16S RNA sequencing. The sequencing results were compared with BLAST on NCBI, and the results showed that MDL511 was... Pediococcus pentosaceus .

[0049] Whole-genome sequencing of MDL511 was performed; the genome map is shown below. Figure 4 The DNA G+C content (mol%) was 37.47%.

[0050] The results of drug resistance gene analysis showed that when the whole genome sequencing results of MDL511 were compared with the CARD drug resistance gene database, the number of possible drug resistance genes matched was 0 when the Identity was greater than 80% and the Coverage was greater than 70%, meaning that there were no drug resistance genes.

[0051] Virulence gene analysis results showed that when the whole genome sequencing results of MDL511 were compared with the VFDB virulence gene database, the number of possible virulence genes matched was 0 when the Identity was greater than 80% and the Coverage was greater than 70%, meaning that no virulence genes were found.

[0052] Analysis of mobile elements revealed four gene islands in the MDL511 genome. However, no virulence or resistance genes were found, indicating an extremely low risk of horizontal metastasis.

[0053] Cultivating and preparing probiotic inoculum: Lactobacillus paracasei MDL511 strain was inoculated into MRS liquid medium at a 2% inoculum and activated by anaerobic culture at 37°C for 16 h. This activation was repeated twice to obtain a seed culture. The seed culture was then inoculated into MRS liquid medium at a 4% inoculum and anaerobic fermented at 37°C for 16 h to obtain the fermentation broth. The fermentation broth was centrifuged and washed to obtain the bacterial cells, which were then mixed with a protectant (m:v) at a 1:4 ratio and freeze-dried to obtain the bacterial agent. The viable count in the bacterial agent was 2 × 10⁻⁶. 11 CFU / g.

[0054] Protectant formula: 150g / L skim milk powder, 100g / L trehalose, 50g / L fructooligosaccharides, 10g / L monosodium glutamate.

[0055] The formula for MRS medium (g / L) is as follows: Peptone 10g / L, beef extract 10g / L, glucose 15g / L, lactose 15g / L, yeast powder 5g / L, diammonium hydrogen citrate 2g / L, K2PO4·3H2O 2.6g / L, MgSO4·7H2O 0.1g / L, MnSO4 0.05g / L, Tween 80 1mL / L, cysteine ​​0.5g / L.

[0056] Example 2

[0057] Experimental Methods: Male C57BL / 6J mice (6–8 weeks old, SPF grade) were used as experimental animals. After purchase, they were housed in the animal facility for a 7-day acclimatization period, during which baseline body weight and food intake were recorded. Subsequently, they were randomly divided into three groups: ① Normal control group (NC, normal diet, PBS administered by gavage, n=6); ② HFD model group (HFD+PBS, n=5); ③ HFD+probiotic group (HFD+1×10⁻⁶ probiotics). 10 CFU / day probiotic, n=6).

[0058] High-fat diets (HFD) provide approximately 60% of their energy (kcal) from fat.

[0059] Intervention strategy: Start with HFD and simultaneously administer probiotics to assess the preventive effect.

[0060] Intervention duration: 6 weeks of HFD; probiotics were administered continuously for 6 weeks.

[0061] Probiotics were administered orally via gavage at a dose of 1×10⁻⁶. 10 CFU / day, volume 200 μL; Suspension preparation: The strain was resuspended in sterile PBS and used on the same day of preparation; the concentration was calibrated using CFU / mL.

[0062] Dosage frequency: once daily at a fixed time (e.g., 10:00 AM) for 6 consecutive weeks.

[0063] The following indicators were tested in the experiment: 1. Weight: Record once a week (within the same time period).

[0064] 2. Food and water intake: Measured once a week per cage (converted to per mouse).

[0065] 3. Sampling was performed 6 weeks after the experiment. Animals were fasted for 12 hours before sampling (unless nighttime metabolic parameters were being measured). Animals were deeply anesthetized with sodium pentobarbital (50–60 mg / kg, intraperitoneal injection), blood was collected via the heart, and the animals were euthanized. All procedures were performed in accordance with animal experimental ethics. Collected blood was allowed to stand at room temperature for 30 minutes, then centrifuged at 3000 rpm for 10 minutes. Serum was separated and stored at -80°C. The obtained serum was used to detect blood biochemistry: lipids (TG, TC, HDL, LDL) and liver function (ALT, AST).

[0066] Histopathological examinations were performed: HE (liver, fat), Oil Red O (frozen sections of liver fat deposition), and adipocyte size measurement (ImageJ).

[0067] Adipose tissue was collected from different parts of mice, and the percentage of body weight of fat in each part of each mouse was calculated.

[0068] Data are expressed as mean ± standard error (Mean ± SEM), with sample sizes n = 6:5:6. Statistical comparisons were performed using one-way ANOVA followed by Tukey's test for multiple comparisons. Statistical significance was set at p < 0.05. In the figures, * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001.

[0069] Experimental results: Line graphs showing changes in body weight in different groups of mice are shown below. Figure 5 As shown in the figure, the comparison chart of mouse weight gain is as follows. Figure 6 As shown.

[0070] The results showed that, compared with HFD mice, MDL511 intake significantly slowed down the rate of weight gain and significantly reduced the amount of weight gain in mice.

[0071] The statistical results of the percentage of epididymal fat, groin fat, and perirenal fat in different groups of mice are shown in the figure below. Figure 7 As shown, the results indicate that, compared with HFD mice, MDL511 intake significantly reduced the percentage of body fat in various parts of the mice.

[0072] The comparison of the levels of four lipid parameters (TC (cholesterol), TG (triglycerides), HDL-C (high-density lipoprotein), and LDL-C (low-density lipoprotein) in different groups of mice is shown in the figure below. Figure 8 As shown, compared with HFD mice, MDL511 intake significantly reduced blood cholesterol (TC) and low-density lipoprotein (LDL-C) levels in mice.

[0073] The comparison of serum ALT and AST levels in mice from different groups is shown in the figure below. Figure 9 As shown, compared with HFD mice, MDL511 intake significantly reduced serum ALT levels.

[0074] HE staining images of epididymal adipose tissue from different groups of mice are shown below. Figure 10 As shown, the results indicate that, compared with HFD mice, MDL511 intake significantly improved the hypertrophy and irregular morphology of adipocytes in the epididymal adipose tissue of mice induced by HFD.

[0075] HE staining images of liver tissue from mice in the same group are shown below. Figure 11 As shown, the results indicated that the liver tissue of HFD mice showed a large number of fat vacuoles and fatty lesions; the intake of MDL511 significantly restored the morphology and structure of the liver tissue.

[0076] Oil Red O staining images of liver tissue from different groups of mice are shown below. Figure 12 As shown, a large number of red lipid droplets accumulated in the liver tissue of HFD mice; intake of MDL511 significantly reduced the lipid droplet level in liver tissue.

[0077] Therefore, the experiments of this invention prove that: MDL511 can significantly reduce weight gain, fat accumulation, serum cholesterol, LDL-C and ALT levels in mice induced by a high-fat diet, and significantly improve liver lesions and fat accumulation caused by HFD. MDL511 has a lipid-lowering effect.

Claims

1. *Pediococcus pentosaceus* MDL511, deposited at the China General Microbiological Culture Collection Center (CGMCC), address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, accession number: CGMCC No. 35201, deposit date: July 11, 2025, strain classification name: *Pediococcus pentosaceus* ( Pediococcus pentosaceus ).

2. A Pediococcus pentosaceus MDL511 inoculum, wherein the active ingredient comprises the fermented cells of Pediococcus pentosaceus MDL511 as described in claim 1.

3. The Pediococcus pentosaceus MDL511 inoculum as described in claim 2, characterized in that... The viable count of the *Pediococcus pentosaceus* MDL511 is not less than 5 × 10⁻⁶. 10 CFU / mL or 2×10 11 CFU / g.

4. The application of Pediococcus pentosaceus MDL511 as described in claim 1 in the preparation of lipid-lowering products.

5. The nutrition as described in claim 4, characterized in that... Application of Pediococcus pentosaceus MDL511 in the preparation of products that lower serum cholesterol, LDL-C, and ALT levels.

6. The use of Pediococcus pentosaceus MDL511 as described in claim 1 in the preparation of products for the prevention, improvement or treatment of obesity.

7. The application as described in claim 5, characterized in that... Application of Pediococcus pentosaceus MDL511 in the preparation of products that reduce weight gain and fat accumulation caused by a high-fat diet.

8. A lipid-lowering product comprising the Pediococcus pentosaceus MDL511 bacterial agent as described in claim 2.

9. A weight loss product comprising the Pediococcus pentosaceus MDL511 bacterial agent as described in claim 2.

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